The present invention relates to novel luciferase having resistance to a surfactant and a method for measuring intracellular ATP using the same.
Intracellular ATP is routinely measured for determining the presence of cells in a sample or the number of cells in the fields of food sanitation, biology, clinical examinations, medical science, ultrapure water, and environmental science. A general method for measuring intracellular ATP comprises the steps of adding an ATP extraction reagent containing a surfactant as an effective component to a sample containing cells, extracting intracellular ATP, adding a luminescence reagent containing luciferase into the sample, and then measuring the total amount of light emitted.
Luciferase is an enzyme that catalyzes luminescence reaction of luciferin, which is a substrate, in the presence of ATP and magnesium ion. Luciferase used in a method for measuring intracellular ATP includes those derived from firefly species, such as GENJI firefly (Luciola cruciata), HEIKE firefly (Luciola lateralis), North American firefly and Russian firefly, etc.
Intracellular ATP can be extracted by adding an ATP extraction reagent to a sample containing cells and then stirring the sample.
To make full use of the capabilities of the extraction reagent, preferably the reaction agent is added so that the concentration of a surfactant becomes 0.05% or more of the mixture of the sample and the extraction reagent. However, a condition where the concentration of the surfactant is 0.05% or more, this inhibits significantly the enzyme reaction in the process of measuring ATP concentration by bioluminescence. Thus the sensitivity and accuracy of measurement are largely impaired. This is because a surfactant at such a high concentration lowers luciferase activity.
For example, North American firefly luciferase activity decreases to about 20% in the presence of 0.1% enzalkonium chloride (See Table 1).
On the other hand, inhibition of the bioluminescent reaction can be reduced with a lower concentration of surfactant. However, in this case the extraction efficiency for ATP would be insufficient.
A method wherein cyclodextrin or its derivative is used is a known method for suppressing the inhibition of luminescence reaction by a surfactant (Japanese Patent Application Laid-Open No. 6-504200).
Among methods for measuring intracellular ATP wherein intracellular ATP is extracted by allowing a sample to contact with a surfactant and subsequently ATP is measured by luciferin-luciferase bioluminescent reaction method, a method for measuring intracellular ATP characterized by the application of the bioluminescent reaction method after allowing a sample, from which ATP is extracted, to contact with cyclodextrin (Japanese Patent Application Laid-Open Publication No. 7-203995) is also known.
There has been no attempt so far to suppress the inhibition of bioluminescent reaction due to a surfactant focusing on luciferase.
The purpose of the invention is to provide a novel luciferase having anti-surfactant resistance, whose activity is not impaired by the presence of a surfactant at a high concentration. The other purpose of the invention is to provide a method, comprising the steps of extracting intracellular ATP using a surfactant and measuring intracellular ATP by bioluminescent reaction using a luciferase, which can lower the inhibition of bioluminescent reaction due to a surfactant without a decrease in efficiency in extracting intracellular ATP.
In the context of this specification, the term xe2x80x9csuppressxe2x80x9d is used to describe significant reduction of the inhibition of the luminescence reaction by a surfactant and the complete elimination of this inhibition.
The present invention relates to a luciferase having anti-surfactant resistance.
The luciferase having resistance to a surfactant includes a luciferase, wherein an amino acid at the 490-position, or an amino acid corresponding to the amino acid at 490-position of GENJI firefly or HEIKE firefly is substituted by an amino acid other than amino acid, e.g., lysine in the amino acid sequence of a wild-type firefly luciferase.
Further, the luciferase having resistance to a surfactant includes a polypeptide consisting of (a) or (b):
(a) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO-4,
(b) A polypeptide comprising additions, deletions, or substitutions of one or more of amino acids in the polypeptide of (a), and having luciferase activity resistant to a surfactant, or
a polypeptide consisting of (a) or (b):
(a) A protein consisting of an amino acid sequence shown in SEQ ID NO:6,
(b) A protein comprising additions, deletions, or substitutions of one or more of amino acids in the polypeptide of (a), and having luciferase activity resistant to a surfactant.
Further, the present invention relates to a luceferase gene encoding the luciferase having resistance to a surfactant.
Furthermore, the present invention relates to a recombinant vector containing the luceiferase gene encoding the luciferase having resistance to a surfactant.
The present invention also relates to a transformant containing the recombinant vector.
In addition, the present invention relates to a method for producing the luciferase, comprising the steps of culturing the recombinant in a medium, and collecting luciferase with resistance to a surfactant from the culture product.
Moreover the present invention relates to a method for measuring intracellular ATP, comprising the steps of a first step wherein ATP is extracted in the presence of a surfactant from cells in a sample; a second step wherein a luminescence reagent containing luciferase is added to the extracted ATP solution so as to cause light emission; and a third step wherein the light emission is measured, and characterized in that luciferase having resistance to a surfactant is used.
This specification encompasses the description and/or drawings given in Japanese Patent Application No. H09-361022.